Automated system for micropropagation and culturing organic material

ABSTRACT

An automated system for growing plant material includes an first length of membrane material having a plurality of open growing chambers and a second length of membrane material having a plurality of growing chambers filled with the plant material. A media preparation unit mixes measured amounts of individual stock solutions for dispensing media into the open growing chambers by a fill unit. A fill check scanner unit determines that a sufficient amount of media has been dispensed within each growing chamber. A sterilization unit sterilizes the media filled open growing chambers which then pass to a cooling and storage unit for cooling and storing the media-filled open growing chambers until ready for planting with plant material. The second length of plant-filled growing chambers are housed in a plant culture room where the plant material is permitted to grow. A growth detection scanner determines the extent of growth of the plant material. Upon the plant material reaching a predetermined growth, the plant-filled growing chambers pass to a surface sterilization unit for surface sterilizing the plant-filled growing chambers. A cutting opens the growing chambers and the plant material is removed. The removed plant material is passed through a cutting unit where the plant material is cut into pieces. Each piece of plant material is then planted into a media-filled open growing chamber from the cooling and storing unit. A heat sealer closes the open end of the newly plant-filled growing chambers. The newly plant-filled growing chambers are then transported back to the culture room. A tractor feed apparatus transports the lengths of growing chambers throughout the automated system and a control system synchronizes and controls the operation of each of the units and tractor feed apparatus.

This is a divisional of copending application(s) Ser. No. 07/278,681filed on 12/1/88 and now U.S. Pat. No. 4,978,505 which is a continuationin part of Ser. No. 207,405 filed June 14, 1988 and now abandoned whichis a continuation in part of Ser. No. 021,408 now U.S. Pat. No.4,908,315.

BACKGROUND OF THE INVENTION

The present invention relates generally to the field of automatedapparatus and processes for micropropagation and culturing organicmaterial. More particularly, the invention relates to automatedapparatus and processes for micropropagation and tissue culturing ofplants. Still more particularly, the invention relates to a new andautomated system for performing micropropagation and tissue culturing ofhorticultural and agricultural plants using integuments.

MICROPROPAGATION AND TISSUE CULTURING

Micropropagation is the process of mass producing new generation plantsfrom a single tissue sample taken from a carefully selected parent plantor cultivar. Micropropagation retains the advantages common to all typesof vegetative propagation, i.e., identity of progeny and the ability topropagate non-seed producing plants, while having the additionaladvantage that only a small piece of tissue from the cultivar or parentplant is required. Micropropagation thus eliminates the disadvantagesassociated with the other forms of vegetative propagation.

Tissue culturing is the process of growing cells in vitro and is used togrow both plant and animal cells. Tissue culturing techniques arecommonly used in the early stages of plant micropropagation processwhere it is desirable to rapidly produce plant cells.

Improvements in tissue culturing techniques also have applicationsbeyond the micropropagation of plants. Essentially the same culturingprocess is used to culture animal and even human tissue, such tissuebeing used in the fields of animal agriculture and human and veterinarymedicine. Culturing of organic material other than plant and animalcells and tissue, such as bacteria, viruses and algeas, is alsoperformed in vitro for both research and commercial purposes.Improvements in the procedures and apparatus used to reproduce andmaintain these organisms would be beneficial, for example, toresearchers and industry who require a large or steady supply of suchmaterial. Further, the automated system of the present invention can beadapted for use with germinating seeds and growing plants therefrom.

DEFICIENCIES IN PRIOR ART MICROPROPAGATION TECHNIQUES

The prior art micropropagation process is described in detail inco-pending U.S. application Ser. No. 021,408, page 5, line 3 throughpage 11, line 9, the entire disclosure of which is hereby incorporatedby reference. Despite the advantages conventional micropropagationtechniques offer the commercial grower, there are problems associatedwith the prior art culturing apparatus and processes. One of the primaryproblems is contamination. Any of a variety of microorganisms, includingviruses, bacteria, fungus, molds, yeast and single cell algae, can ruinthe cultures during any of the various stages of micropropagation.

The prior at sterilized glass or plastic culture containers such as testtubes, flasks or bottles have serious drawbacks. For example, sinceplants require both carbon dioxide and oxygen to live and grow, thesecontainers must provide a means for gas exchange. The walls of thesetraditional glass and plastic containers, however, do not permit therequired gaseous interchange. Thus, rubber stoppers having cottonpacking or some similar filter material, loosely fitting caps, orbaffled plastic caps have been employed to allow an adequate exchange ofgas between the tissue or plant and the ambient atmosphere andenvironment. However, such devices restrict the amount and rate of gaswhich can be exchanged. Further, such caps and stoppers do not totallyprotect the plant from contamination by microorganisms such as viruses,bacteria and fungi. Thus, it has been of paramount importance that thetissue culture room and laboratory be maintained under asepticconditions, i.e. kept extremely clean and their atmospheres entirelyfiltered. Further, precise temperature, humidity, and light conditionsmust also be maintained in the culture room when using traditionalmicropropagation techniques and apparatus. Gas exchange is also requiredfor culturing animal cells and for certain other microorganisms.Traditional flasks, petrie dishes and the like, while allowing for acertain degree of gas exchange, also allow contamination to occur.

The original cost of the traditional glass or plastic culturecontainers; the labor and equipment cost to maintain the sterility ofthe containers; and the added cost of the facilities, equipment, andrelated conditions required to maintain a sterile growing environment,all represent major cost factors associated with the use of suchcontainer in conventional culturing processes.

A further significant disadvantage of the prior art micropropagationprocess and apparatus is the fact that the conventional culturingcontainers do not lend themselves to use in an automated system.Currently, each step of the micropropagation process must be performedby time consuming and laborious manual operations. For example, when atissue sample which has survived stage one and has grown to a size thatit is ready for multiplication, the culture container, a glass testtube, for example, must be carried from the culture room to thelaboratory and placed under a laminar flow hood. There, a techniciansitting in front of the hood, will typically spray the container with asolution of alcohol to kill microorganisms which might be on or near theentrance of the container and contaminate the culture during the tissuemanipulation. Next, the technician must grasp the test tube in one hand,remove the cotton filled rubber stopper (in this example), remove thetissue sample with sterilized forceps and place it on a sterilizedworking surface. The technician must then cut the tissue sample into anumber of individual samples each of which will then be placed in asterile container with fresh media.

The containers and media to be used in this next stage will themselveshave already been manually prepared. Typically, a measured amount ofprepared media is placed in each test tube, with the test tubes beingheld vertically in a conventional test tube rack. The racks ofmedia-filled test tubes are then sterilized and transferred to thelaminar hood for the technician's use in the next tissue manipulation.Similarly, culture container lids and stoppers must also be cleaned,sterilized and placed under the laminar flow hood for the technician'suse. Once cooled, the technician will grasp a clean and sterilized testtube in one hand and will insert one portion of the newly divided tissuesample into the sterilized media with the other hand, and then place acotton filled and sterilized stopper on the test tube and replace thetest tube in the rack. Once the tissue manipulations are completed, theracks containing the new cultures are then transported back to theculture room.

As can be appreciated, the number of cultures which can be produced isdirectly related to the efforts and abilities of the technicians andmore particularly to the manual dexterity of the technicians.Furthermore, the extensive manual operation and human involvement in theprocess creates a tremendous potential for contamination, even despitethe precautions currently taken, such as requiring the technicians towear surgical gloves and masks.

Additionally, the remaining steps in the micropropagation process mustbe carried out manually. Test tubes are manually loaded and unloadedinto washing apparatus and frequently require a manual washing tocompletely remove media or residue from a container which had acontaminated culture. Likewise, it is time consuming to manually mixmedias and fill the test tubes or culture containers with the preparedmedia in measured quantities. Culture vessels or containers are alsomanually loaded into autoclaves for sterilization. As explained above,before opening a culture container, its sides are typically manuallysprayed with a solution of alcohol or chlorine solution to killmicroorganisms which might contaminate the culture once the container isopened.

It is also currently left to technicians to visually inspect the growingcultures for signs of contamination and growth and take the appropriateaction depending upon their observation. For example, when tissue orplantlets have reached their desired size, technicians must manuallytransfer the culture containers from the culture room to the laboratoryin order to perform the next manipulation. When a culture iscontaminated, it is also manually removed from the culture room andtransported to a station for disposal and for container cleaning andsterilization.

Current micropropagation techniques also lack the ability to monitorinventory through automatic means. Instead, inventories are controlledby maintaining physical separation between the cultures of the variousplants being grown and by simply counting the number of culturecontainers and the cultures contained therein.

As can be appreciated, the conventional micropropagation process isextremely labor intensive and costly. In addition, the level ofproduction is limited by the number and abilities of the techniciansinvolved. A well qualified technician, using conventional culturingapparatus and procedures can establish approximately 350 cultures perday. Using a laminar flow hood to its maximum efficiency by employingthree technicians, each working eight hours in a 24 hour day, themaximum number of cultures which can be established by well trainedtechnicians in a day is approximately 1050. Accordingly, there is a needin the art for an automated system for performing micropropagation andthe culturing of organic material. It is desirable that such a systemeliminate the time consuming and extremely expensive manual stepscurrently employed, including tissue manipulation, container cleaningand sterilization, culture transportation, media preparation, andfilling. In addition, it is desirable that such a system have thecapability of automatically detecting culture containers which have beenunfilled or underfilled with media, cultures which have becomecontaminated, and tissue samples of plantlets which are ready for thenext stage of micropropagation. An automated system also having thecapability of tracking a culture throughout the micropropagation processand automatically computing the inventory of the various plants ormaterials being cultured would also be a great advance over thetraditional culturing apparatus and processes.

Other objects and advantages of the invention will appear from thefollowing description.

SUMMARY OF THE INVENTION

The automated system for growing plant material includes a length ofmembrane material having a plurality of open growing chambers. Thepreferred membrane material is a high density polyethylene sealedtogether at predetermined locations to form a plurality of growingchambers having an open end for the insertion of media and plantmaterial. A media preparation unit mixes measured amounts of individualstock solutions to prepare a selected growing media for the plantmaterial. A fill unit dispenses the media into the open growing chambersof the length of membrane material. A fill check scanner unit scans themedia-filled open growing chambers to insure that each of the growingchambers has been filled with a predetermined amount of media. Themedia-filled growing chambers then pass to a sterilization unit forsterilization. A cooling and storage unit cools and stores themedia-filled open growing chambers until it is time for the insertion ofplant material.

Sealed growing chambers, previously filled with media and plantmaterial, are housed in a plant culture room where the plant materialhas been permitted to grow in another length of membrane material. Thelength of plant-filled growing chambers is periodically passed through agrowth detection scanner unit to scan the plant material to determinethe extent of plant growth. Upon the plant material having reachedsufficient growth, the length of plant-filled growing chambers istransported from the culture room to a surface sterilization unit forsurface sterilizing the exterior of the growing chambers. A cutting unitopens the plant-filled growing chambers in preparation for the removalof the plant material. The plant material is removed from theplant-filled growing chambers by the injection of sterilized water intothe closed end of the growing chamber to wash the plant material out ofthe opposite open end of the growing chamber which had been opened bythe cutting unit. A rotating tissue containment device receives theplant material for transporting the plant material to the plant cuttingunit. The plant material is extracted from the tissue containment unitand pushed against a reciprocating blade which cuts the plant materialinto individual pieces. A planting unit inserts individual pieces of thecut plant material into the media-filled open growing chamberspreviously stored in the cooling and storage unit. After themedia-filled open growing chambers have been planted with a piece ofplant material, the open end of the growing chambers is closed by heatsealing. The newly plant-filled growing chambers are then transportedback to the culture room for new growth.

A tractor feed apparatus transports the lengths of growing chambersthroughout the automated system. A control system synchronizes andcontrols the timing, sequence and operation of each of the units andtractor feed apparatus. Bar coding units uniquely identify each growingchamber to track each growing chamber as it progresses through theoperations of the automated system.

BRIEF DESCRIPTION OF THE DRAWINGS

For a detailed description of a preferred embodiment of the invention,reference will now be made to the accompanying drawings, wherein:

FIG. 1 is a schematic of the automated system of the present inventionfor automating the micropropagation and tissue culturing of organicmaterial;

FIG. 2 is a perspective view of a roll of a continuous length ofcellules for the automated system of FIG. 1;

FIG. 3 is a fragmented view of a portion of the continuous length ofcellules of FIG. 2;

FIG. 4 is a cross-section of the continuous length through a cellule atplane 4--4 in FIG. 3;

FIG. 5 is a plan view of the mechanism to manufacture the continuouslength of cellules from a film;

FIG. 6 is a partial plan view of a portion of the tractor feed apparatusfor moving the continuous length of cellules throughout the automatedsystem of FIG. 1;

FIG. 6A is a perspective view of a portion of the tractor feed apparatusof FIG. 6;

FIG. 7 is a cross section of the tractor feed apparatus at plane 7--7 ofFIG. 6;

FIG. 8 is a schematic of the media preparation and media fill unitsshown in FIG. 1;

FIG. 9 is an elevation view of the media fill apparatus of FIG. 8;

FIG. 10 is a perspective view of a portion of the media fill apparatusof FIG. 9;

FIG. 11 is a top view of the fill check scanner of FIG. 1;

FIG. 12 is an elevation view of the sterilization unit of the automatedsystem of FIG. 1;

FIG. 13 is a sectional view of the sterilization unit taken at plane13--13 in FIG. 12;

FIG. 14 is a sectional view of the sterilization unit taken at plane14--14 in FIG. 12;

FIG. 15 is an enlarged view of the tractor feed apparatus disposedwithin the sterilization unit shown in FIG. 14;

FIG. 16 is a perspective view, partly in section, of the cooling andstorage unit of the automated system of FIG. 1;

FIG. 16A is a section view of the cooling and storage unit taken atplane 16A--16A in FIG. 16;

FIG. 17 is a sectional elevation view, partly diagrammatical, of thesurface sterilization unit of the automated system of FIG. 1;

FIG. 18 is a sectional view of the tractor feed apparatus disposedwithin the surface sterilization unit taken at plane 18--18 of FIG. 17;

FIG. 18A is a sectional view of another portion of the surfacesterilization unit taken at plane 18A--18A of FIG. 17;

FIG. 19 is a perspective view of a portion of the tissue manipulationunit of the automated system of FIG. 1;

FIG. 20 is a front view of the tissue manipulation unit of FIG. 19;

FIG. 20A is a front sectional view of the tissue manipulation unit ofFIG. 20 with the extraction member in the staged position;

FIG. 20B is a side sectional view of the tissue manipulation unit ofFIG. 20 with the extraction member in the extended position;

FIG. 21 is a side sectional view of the tissue manipulation unit of FIG.19;

FIG. 21A is a side sectional view of the tissue manipulation unit ofFIG. 21 with the cutting blade and stuffing mechanism in the stagedposition;

FIG. 21B is a side sectional view of the tissue manipulation unit ofFIG. 21 with the cutting blade and stuffing mechanism in the extendedposition;

FIG. 22 is a perspective view, partially in section, of the culture roomof the automated system of FIG. 1; and

FIG. 23 is a block diagram of a control system for the automated system.

DESCRIPTION OF THE PREFERRED EMBODIMENT System 10 Overview

Referring initially to FIG. 1, there is shown a schematic illustrationof the automated apparatus 10 for performing micropropagation and tissueculturing of plant tissue. The apparatus of the present invention isemployed to automatically perform micropropagation and tissue culturingprocedures through the use of the integument, a new pliable growingcontainer described in co-pending applications Ser. No. 207,405 filedJune 14, 1988 and Ser. No. 021,408 filed Mar. 4, 1987, both incorporatedherein by reference.

In general, an integument is a growing or culture container formed froma translucent membrane that is liquid and contaminant impermeable, butwhich allows necessary gas exchange and light transmission between theliving tissue being cultured and the ambient environment. The membraneis formed into an envelope or cellule for containing the tissue andgrowth medium. Once the tissue and growth medium are placed in thecellule of the integument, the cellule is sealed and thus closed to theambient environment. As described in co-pending applications Ser. No.207,405 filed June 14, 1988 and Ser. No. 021,408 filed Mar. 4, 1987, anintegument pack includes a number of individual cellules which arepliant and collapsible such that they may be rolled. For use with theapparatus and method of the present invention, it is preferred that anintegument roll 20, described in more detail below, be employed.Integument roll 20 comprises a plurality of integuments 22 attached atadjacent edges forming a continuous ribbon-like sheet or length 24 ofinteguments 22 loosely rolled onto a spool 26.

Referring again to FIG. 1, the integument roll 20 is housed in anintegument storage unit 28. In operation, cellules 30 of integuments 22from integument roll 20 are transported as a continuous length 24 by atractor feed mechanism 50 throughout the automated system 10. Afterleaving storage unit 28, the cellules 30 move, first to the media fillapparatus 70. A media preparation unit 80 automatically mixes theingredients and proportions thereof needed to form the growth mediumused for particular plants in the various stages of micropropagation.Once the cellules 30 are appropriately positioned within the media fillapparatus 70, media fill apparatus 70 injects media 92 from mediapreparation unit 80 into the individual cellules 30 of the continuouslength 24 of integuments 22 as it is unrolled from the integument roll20. Once filled with the measured quantity of the growth medium 92, abar code indicating the type media is placed on the outside surface ofcellules 30 by bar coding means 93. Cellules 30 are then transported toa fill-check scanner 90 to insure the appropriate amount of growthmedium 92 has been inserted into the cellules 30. The cellules 30 withgrowth medium 92 are then transported to the sterilization unit 100where they are heated under pressure to kill any microorganisms in or onthe cellules 30 or the prepared media 92. From the sterilization unit100, the sterilized cellules are transported to the cooling and storageunit 110. The sterilized cellules with media are stored in the coolingand storage unit 110 until plant tissue growing in other cellules, ashereinafter described, are ready for transplanting into the sterilizedcellules stored in unit 110. For transplanting, the cellules aretransported from unit 110 on to the tissue manipulation unit 120.

All the required tissue manipulations of the micropropagation processare carried out within the tissue manipulation unit 120. The sterilizedcellules and growth media are therein invested with tissue samples 122and then closed by heat sealing in sealing unit 310 to preventcontamination. The invested cellules are then coded by bar coding means311 with a bar code indicating the type of plant and the date theculture was established.

The coded cellules 30 with plant tissue are then transported to andthrough the culture room 130 where they are exposed to a growingenvironment conducive to the particular variety of plant being grown andthe stage of micropropagation. After the culture has been in the cultureroom 130 for the appropriate time period and grown to the desired stageof development, the cellules 30 containing the cultures are transportedthrough a growth detection scanner 140, which detects the growth of theplant material or tissue, and through a bar code reader 141. If theculture is ready for the next stage of micropropagation, the cellulescontaining the cultures are transported back into the tissuemanipulation unit 120 where the cellules are surface sterilized andwashed in surface sterilization unit 320 and opened in cellule cuttingunit 280. The tissue samples 122 are then removed and cut into smallertissue samples for transplanting or investment in new sterile celluleswith growth media from cooling and storage unit 110 in tissue plantingunit 290. The cellules with new tissue samples are then sealed insealing unit 310 and transported back into the culture room 130. Oncethe appropriate number of tissue multiplications have been performed andthe desired number of plantlets have been produced, the cellulescontaining the plantlets are transported from the culture room 130 tothe packaging system 160 where the still sealed cellules are boxed forshipping.

The entire process is controlled and monitored by control system 150.

Integument Roll 20

Referring now to FIG. 2, there is shown an integument roll 20 generallycomprising a continuous ribbon-like sheet or length 24 of individualcellules 30 wrapped loosely around a spool 26. One portion of thecontinuous length 24 of cellules 30 is depicted in FIG. 3 and, asdepicted, comprises individual cellules 30a, 30b, and 30c. As best shownin FIG. 4, an individual cellule 30 is formed by a front membrane 32 andback membrane 34 which are attached at their lower extremities by a wideheat-sealed lower band 36. It is preferred that lower band 36 beapproximately one-half inch wide. While a narrower heat seal willsuffice to prevent contamination, the wider heat-sealed band adds anextra measure of protection against the introduction of microorganismsand allows for lower tractor perforations 42 in lower band 36 which, asdescribed below, are used in conjunction with the tractor feed apparatus50 shown in FIGS. 6 and 7.

Referring still to FIGS. 3 and 4, front membrane 32 and back membrane 34are also heat sealed along lines perpendicular to band 36 as shown at38a, 38b, 38c, and 38d, thereby forming individual cellules 30a, 30b,and 30c. Preferably, heat seals 38 do not extend the entire width ofmembranes 32 and 34, but instead stop approximately one-half inch shortof the upper edges of membranes 32 and 34, thereby leaving upper frontand back bands 46 and 48 respectively, unattached. In thisconfiguration, cellule 30 is defined by heat seals 38 and lower heatseal band 36 leaving initially an open end 52 which serves as an entryport into cellule 30 for receiving plant tissue and growth media. Lowertractor perforations or apertures 42 are formed in lower heat seal band36 and upper tractor perforations 44, such a 44a and 44b, are formed inupper bands 46 and 48 at uniform distances along the entire continuouslength 24 of integument roll 20.

Referring now to FIG. 5, there is shown a manufacturing process for theintegument roll 20. Although it is anticipated that the integument 22will be manufactured separately from the micropropagation process, themanufacturing process may be a part of the automated system 10. Theintegument roll 20 is manufactured by the melt blowing of a polyolefinfilm such as polyethylene. In manufacture, the film is blown into alarge bubble which is drawn upward to obtain the desired film thicknessand is then cooled. The blown film is then drawn between rollers where acontinuous double layer of film 54 is drawn from the film makingmachine. In-line operations can then be made on the double layer of film54. For example, two integument rolls 20a and 20b can be manufacturedfrom the double layer of film 54. As the double layer of film 54 isdrawn from the film making machine or roll 56, it may be drawn over aheat sealing roller 58 as shown in FIG. 5. Heat sealing roller 58includes raised portions 60 and 62 used to simultaneously form heat sealband 36 and heat seals 38 respectively on two continuous lengths 24a and24b which, at this point, are joined at their upper edges 64. Afterpassing over heat sealing roller 58, the double layer of film 54 maypass over perforation rollers 66 which include projections 68 formedabout their circumference. Projections 68 engage recesses formed in amating rollers (not shown) which are positioned above perforationrollers 66. As the double layer of film 54 is passed between theserollers, bands 36, 46 and 48, best shown in FIG. 4, are all perforated.The double layer of film 54 is then cut into two separate continuouslengths 24a and 24b as the film 54 is passed through a stationary knifeblade 72. The two lengths 24a and 24b are then wound on spools 26a and26b.

The polymeric material for cellules 30 is critical to providing thenecessary environment for housing plant and animal life. In particular,it is important to achieve optimum gas exchange and light transmissionto permit the necessary biochemical activity conducive to life. Thematerial must readily pass oxygen and carbon dioxide between the ambientatmosphere and the cellule 30 for use by the contained plant or animallife in their metabolic processes to preserve the organic material andthe like in a living condition. Thus, the cellule 30 is made of asemi-permeable and translucent material which permits gas transfertherethrough. The preferred material for cellule 30 is a polyethylenefilm from 1.0 to 2.0 mils. thick. It is preferred that the material havethickness of 1.25 mils. If the membrane material is thinner than 1.0mil, handling the cellule 30, and especially opening cellule 30, is mademore difficult because the opposing sides 32, 34 of the material ofcellule 30 tends to adhere to each other when formed in such thin filmsless than 1.0 mil. Although a translucent low density polyethylene issuitable and even allows greater gas permeability, a high densitypolyethylene is preferred. The high density polyethylene can withstandgreater extremes in temperature, such as is encountered in an autoclave,where a low density polyethylene may tend to melt, distend, or distort.Other polymeric materials may be used where the gas and water vaportransmission rates are comparable to that of the present invention.

The gas transmission rates of the material for cellule 30 is of theutmost importance. For practicing the invention described herein, it ispreferred that the membrane material have a permeability to CO₂ of from200 to 1190 cc/100 sq. in/24 hours at 1 atm. and a permeability to O₂ offrom 100 to 400 cc/100 sq. in/24 hours at 1 atm. Another importantfactor may be the moisture vapor transmission rate which is preferredfrom 0.2 to 0.684 gm/100 sq. in/24 hours at 1 atm. The preferred highdensity polyethylene film exhibiting the above characteristics is highdensity polyethylene material no. HiD-9650 manufactured by ChevronChemical Company of Orange, Texas. Upon sealing the cellule 30, theorganic material is completely enveloped and enclosed from the ambientatmosphere and environment so as to prevent any introduction ofcontaminants and permit the necessary gas exchange between the organicmaterial therein and the atmosphere of the ambient environment. Thematerial of cellule 30 is also translucent to enable the organicmaterial to receive the necessary light for life and growth.

The published specifications for high density polyethylene HiD-9650 aremelt index of 0.3 (gms/10 min); density 0.950 (gms/cc); dart impact of90 (gms/mil at 26 inches); tensile strength at break of 7400 (psi);elongation of 4 and 60%; Elmendorf tear md/td of 16/400 (gms/mil); and amoisture vapor transmission rate of 0.35 (gms/100 sq. in. 24 hr./mil).

Tractor Feed Apparatus

The tractor feed apparatus 50 operates to transport the continuouslength 24 of cellules 30 throughout and between each of the apparatuswhich comprises the automated system 10. Tractor feed mechanism 50comprises a plurality of individual tractor feed belts, belt guidechannels, supports, rollers and drive motors as described in more detailbelow. The description of one segment of the tractor feed apparatus willtypify the remaining segments of the mechanism.

Referring now to FIGS. 6 and 7, there is shown one portion of thetractor feed apparatus 50. Depicted in FIG. 6 is a partial plan view ofthat portion of the tractor feed apparatus 50 which serves to draw thecontinuous length 24 of cellules 30 from roll 20 mounted in theintegument storage unit 28 and to transport the cellules 30 ofintegument roll 20 to the media fill apparatus 70 shown in FIG. 1. Asshown in FIGS. 6 and 7, the tractor feed apparatus 50 generallycomprises driver and receiver support plates 74, 76, studded drive belts78, receiving belts 82, driver and receiver belt guide channels 84, 86,drive and receiver rollers 88, 89 and tensioning rollers 94respectively. Belt guide channels 84, 86 are attached to support plates74, 76 which are themselves anchored to a supporting base, not shown,attached or resting on the floor or suspended from the ceiling or othersuitable support structure. It is preferred that guide channels 84, 86be bolted to support plates 74, 76. In this manner, the distance betweenupper guide channels 84a, 86a and lower guide channels 84b, 86b canreadily be changed in the event that an integument roll 20 having adifferent dimension is later used. Studded drive belts 78 and receivingbelts 82 are received by and travel within the recesses of driver andreceiver belt guide channels 84, 86 respectively. It is preferred thatdriver and receiver belts be made of Teflon. Motion is imparted to thebelts 78, 82 by drive and receiver rollers 88, 89, shown in FIG. 6,located at the ends of driver and receiver guide channel 84, 86. Asshown in FIG. 7, driver and receiver rollers 88, 89 extend through slots75 formed in support plates 74, 76. Referring to FIG. 6A, driver andreceiver rollers 88, 89 include teeth 98 which mesh with indentations102 on the inner surface 104 of drive and receiver belts 78, 82. Theengagement of teeth 98 with indentations 102 prevents slippage betweenrollers 88, 89 and belts 78, 82. Upper and lower drive rollers 88a, 88bare mounted on a driver shaft 106 and upper and lower receiver rollers89a, 89b are mounted on a receiver shaft 108. Shafts 106, 108 arerotatably supported by journal bearings 118 and are driven by a commonmotor 112 through gears 107a, 107b. It should be appreciated that it mayonly be necessary to drive one of the shafts 106, 108 using one gear 107driven by motor 112. Journal bearings 118 are mounted on belt guidechannels 84, 86.

As rollers 88, 89 are rotated clockwise and counterclockwiserespectively, as viewed in FIG. 6 projections 114 on studded drive belts78 mate with indentations 116 formed in receiving belts 82. Theprojections 114 and indentations 116 are positioned along the belts 78and 82 so as to coincide with the dimensions between adjacent upper andlower tractor perforations 44, 42 formed in continuous length 24 asshown in FIG. 3. Thus, the lower band 36 and the upper bands 46, 48 oflength 24 are captured and attached between driver and receiver belts78, 82. The guide channels 84, 86 extend between each apparatus in theautomated system 10 as shown in FIG. 1 and thereby transport the length24 throughout the system 10. Tensioning rollers 94 are positioned alongguide channels 84, 86 to tension and guide the belts 78, 82. Althoughnot shown, additional drive and receiver rollers 88, 89 arestrategically positioned between apparatus located throughout theautomated system 10 along guide channels 84, 86 to propel the length 24of cellules to each of the apparatus in the system 10. It should beappreciated that other means may be adapted for attaching the bands to amoving track for transporting the length 24 throughout the automatedsystem 10.

As shown in FIG. 7, continuous length 24 is transported by its upper andlower edges by a total of four belts: upper drive belt 78a; upperreceiving belt 82a; lower drive belt 78b; and lower receiving belt 82b.To ensure that the tractor feed apparatus 50 functions properly and thatcontinuous length 24 of cellules 30 is not damaged from engagement withbelts moving at a number of different velocities, it is important thatdrive rollers 88a, 88b and receiver rollers 89a, 89b be driven at theidentical velocity. As is evident, as rollers 88, 89 are rotatedclockwise and counterclockwise respectively as shown in FIG. 6, thecellules 30 of continuous length 24 are transported along with themoving belts 78, 82 at a uniform velocity. It should be understoodhowever that other continuous lengths 24 may be transported at adifferent uniform velocity depending upon its location in system 10.

Media Preparation Unit 80

As shown schematically in FIG. 8, media preparation unit 80 comprisesmix tank 124, stirrer 126, stirrer motor 128, heater 132, stock solutionrefrigeration unit 134, stock solution containers 136 and metering pumps138. A plurality of stock solution containers 136 are refrigeratedwithin refrigeration unit 134 and maintained at a temperature ofapproximately two degrees centigrade. The stock solution containers 136each contain a separate ingredient or nutrient used in the preparationof the various media used in the micropropagation process. Each mediumused in the process is mixed in a batch mode within the mix tank 124,which is preferably made of stainless steel. When a level switch 142within mix tank 124 signals controller 150 that another batch of mediais required, controller 150 will signal appropriate metering pumps 138,which are in fluid communication with fill lines 144, to inject aprogrammed amount of stock solution through individual fill lines 144extending into mix tank 124. It is preferred that metering pump 138 be aparastolic pump such as Model No. 2P3O4 manufactured by the Mec-O-MaticCo. Such pumps are reliable and extremely accurate. Controller 150 alsoactuates solenoid valve 133 in sterile water line 143 allowing theappropriate amount of sterile water to flow into mix tank 124.

Once the appropriate stock solutions and sterile water have beeninjected into the mix tank 124, the ingredients are heated by heater 132while the solution is stirred by stirrer 126. Stirrer 126 includes animpeller 146 mounted on the end of a shaft 148 which is connected to androtated by stirrer motor 128. Both heater 132 and stirrer motor 128 areactuated by controller 150. The media is stirred and heated to atemperature of approximately 100° C. in order to melt the agar or othergelling agent which is used in the particular growth medium beingprepared. Optionally, the media may be supplemented with nutrients andplant growth regulators. Once the growth medium is prepared, heater 132and stirrer 124 are turned off and the growth medium is then ready forinjection into cellules 30.

Unused media and liquids used to clean and rinse mix tank 124 may bedrained from mix tank 124 through drain 141 and pumped to a disposaltank (not shown).

Media Fill Station 70

Referring now to FIGS. 8, 9 and 10, the media fill station 70 comprisesfill lines 152, parastolic fill pumps 154, injection nozzles 156, nozzletransport rack 158 and filling guide 162. In general, measured amountsof growth medium from media preparation unit 80 are simultaneouslyinjected into a plurality of cellules 30 of continuous length 24 bymedia fill apparatus 70 as shown in FIG. 9.

Five fill lines 152 are in fluid communication with mix tank 124 asshown in FIG. 8 and are comprised of a flexible plastic tubing having aninternal diameter of approximately 5/8 inches. Injection nozzles 156 areconnected to the ends of fill lines 152 and are positioned above fillingguide 162 on nozzle transport rack 158. Nozzles 156 are tapered andsized to be inserted between upper bands 46, 48 of cellules 30 for entrythrough entry port 52 into the chamber of cellule 30. Filling guide 162is supported by support arms 164. As can be seen in FIG. 9, the leadingand trailing edges of filling guide 162 are formed with wedge-shapedends 166 to separate bands 46, 48 of cellules 30. In response to asignal from controller 150 after the unfilled cellules 30 have beenpositioned below filling guide 162, pneumatic cylinders 172 actuate andlower pistons 174, thereby lowering nozzles 156 into position forfilling cellules 30 with growth medium 92. An individual parastolic fillpump 154 is dedicated to each fill line 152 and, like metering pump 138described above, may be Mec-O-Matic Model No. 2P3O4. Upon receipt of asignal from controller 150, fill pump 154 will pump a predeterminedmeasure of mixed growth medium 92 from mix tank 124 through fill lines152, injection nozzle 156 and into cellules 30.

In operation, the continuous length 24 of cellules 30 is drawn bytractor feed apparatus 50 to a media fill station 70 where the upperbands 46, 48 of cellules 30 are separated by filling guide 162 ascontinuous length 24 is drawn by tractor feed apparatus 50 beneathnozzle transport rack 158. Controller 150 actuates drive motors 112positioned along the tractor feed apparatus 50, as previously describedand shown in FIGS. 6 and 7, in timed intervals such that five cellules30 are positioned and remain stationary underneath filling guide 162 forapproximately three seconds while growth medium 92 is injected into thecellules 30. Once in position, controller 150 signals the pair ofpneumatic cylinders 172 to lower nozzle transport rack 158 and injectionnozzles 156. In this manner, nozzles 156 are lowered into the fillingguide 162. Controller 150 then signals the parastolic fill pumps 154 toinject the appropriate measure of media 92 into each of the fivecellules 30. Controller 150 then actuates the pneumatic cylinders 172 toraise injection nozzles 156 back into position shown in FIG. 9 abovefilling guide 162 and then signals drive motors 112 operating tractorfeed apparatus 50 to transport five new unfilled cellules 30 intoposition underneath rack 158 for filling with growth media.

After a cellule 30 has been filled with media and passed through themedia fill station, the cellule may be marked with a bar code by a barcode printing system 93, such as a "Digimark" variable information lasermarker manufactured by Videojet Systems International, Inc. of Elk GroveVillage, Ill. The cellules may include a solid ink mark, portions ofwhich are vaporized by the laser printer of the bar code printing system93 to indicate the type of media in the cellule. Other indicia may alsobe coded on the cellule.

Fill Check Scanner 90

Referring now to FIG. 11, fill check scanner 90 is used to determinewhether the cellules 30 have been injected with the appropriate measureof growth medium 92. Fill check scanner 90 generally comprises enclosure176, light source 178, polarized panel 180 and photo receptor panel 182.As shown in FIG. 11, tractor feed apparatus 50, upon input fromcontroller 150, draws continuous length 24 through the interior ofenclosure 176 so as to transport the media-filled cellules 30 into theenclosure 176 for scanning. As described above with respect to the mediafilling station 70, the cooperation of controller 150, with the drivemotors 112 of the tractor feed apparatus 50 (FIGS. 6 and 7), willtransport cellules 30 for scanning in groups of five. Positioned on oneside of enclosure 176 is a light source 178 which may be, for example,quartz-halogen. Polarized panel 180 is affixed within enclosure 176 asshown in FIG. 11 and divides the interior of the enclosure into twocompartments. Polarized panel 180 is selected so that light wavespassing in a direction perpendicular to the panel 180 will be passedthrough the polarized panel 180; however, light rays traveling in otherdirections will not pass through polarized panel 180.

Light waves which pass through polarized panel 180 will continue throughthe cellules 30 of continuous length 24 and will contact photoreceptorpanel 182 on the opposite side of the enclosure 176. Photoreceptor panel182 comprises a surface containing hundreds of photosensitive cells (notshown). Light waves will pass through the membranes 32 and 34 ofcellules 30 and activate the photosensitive cells on photoreceptor panel182. Light waves penetrating the areas of the cellules 30 filled withgrowth media 92 will be defracted to a greater degree than those whichpass through the portion of cellule 30 containing no media. Accordingly,the light intensity sensed by the portion of photoreceptor panel 182which is directly behind the media-filled portions of the cellules 30will be less than the intensity sensed by remaining portions of panel182. The photosensitive cells on photoreceptor panel 182 areelectrically connected to the controller 150 by a plurality of signalwires 184. In this manner, it can be determined which cellules 30 havebeen filled and whether they have been filled with the appropriatevolume of growth medium 92.

An alternative embodiment of fill check scanner 90 is the "Smarteye"photoelectric sensor manufactured by the Tri-Tronics Company, Inc. ofTampa, Fla. The "Smarteye" photoelectric sensor can sense size, texture,distance, opacity, depth and color so as to have the capability ofdetermining whether an appropriate measure of growth media 92 has beeninjected into a particular cellule.

Upon fill check scanner 90 identifying a cellule which has an inadequateamount of media, the inadequate cellule is marked by an ink jet printersuch as the "Excel" small character ink jet printer 91 manufactured byVideojet Systems International, Inc. of Elk Grove Village, Ill. A printregistration scanner 95, such as the "Smarteye" color mark registrationscanner manufactured by Tri-Tronics Company, Inc. of Tampa, Fla., willsubsequently identify the inadequate cellule by scanning for the inkmark prior to the insertion of plant material in tissue planting unit290. The print registration scanner 95 will send a signal to controller150 which will in turn cause the tractor feed apparatus 50 to pass theinadequate cellule through the tissue planting unit without insertingany plant tissue.

Sterilization Unit 100

Referring now to FIGS. 12 and 13, there is shown sterilization unit 100generally comprising an autoclave 186 used to sterilize the media-filledcellules 30 before the cellules 30 are invested with tissue. Theautoclave 186 comprises a generally cylindrical enclosure 188 mounted ona support structure 190. The enclosure 188 comprises a pressure chamber192 and a closure 194 coaxially aligned and attached by four pneumaticcylinders 196 used to open and close the closure 194. The pressurechamber 192 has attached to its interior entrance an inner lip 198 whichextends around the entire periphery of the interior entrance of thepressure chamber 192 and serves to guide the closure 194 during theclosing of the autoclave 186 by pneumatic cylinders 196. Lip 198 alsoserves to protect an O-ring seal (not shown) from the gases and extremeheat generated during the sterilization procedure. Pressure chamber 192,closure 194 and inner lip 198 are all manufactured from stainless steel,have an inner jacket of monel and have a total thickness of less than1/2 inch.

In operation, the leading end of continuous length 24 is passed througha cutter 202 mounted on the side of autoclave 186 and in cooperationwith the tractor feed apparatus 50. The cutter 202 acts as a guide forthe continuous length 24 of cellules 30 as the cellules 30 are disposedwithin the autoclave 186. Cutter 202 also includes a blade 204 which isactivated by controller 150 after the autoclave 186 has been filled witha strip 200 of cellules 30, strip 200 having a length of as much asseveral hundred feet and comprising many thousands of cellules 30.Referring to FIGS. 13, 14, and 15, integument strip 200 is automaticallyloaded into the autoclave 186 for sterilization by internalloader/unloader apparatus 210 which operates identically to the tractorfeed apparatus 50 previously described. As described in greater detailbelow, the internal loader/unloader apparatus 210 supports andtransports the integument strip 200 by use of a series of drive beltswhich cooperatively engage upper apertures 44, 42 formed in the upperbands 46, 48 and lower band 36 of integument strip 200. The drive beltsare supported in a multi-level serpentine configuration within theautoclave 186 so as to achieve the greatest density of cellules 30 aspossible.

There is shown in FIG. 13 a section view of the autoclave 186 whichschematically illustrates the path of integument strip 200 as it isloaded in serpentine fashion into the autoclave 186. FIGS. 14 and 15depict how the integument strip 200 is supported and transported withinautoclave 186. Referring now to FIGS. 14 and 15, perforated supportplates 214 are rigidly attached to the upper interior surface ofpressure chamber 192. Support plates 214 are perforated so as to enablesteam to penetrate throughout enclosure 188. Attached to the perforatedplates 214 are belt guide channels 216. Retained within belt guideschannels 216 are the drive belts including studded drive belt 218 andreceiving belt 220. As described previously with respect to the tractorfeed apparatus 50, the projections 222 on studded drive belt 218 and theindentations 224 on receiving belt 220 are spaced apart on belts 218 and220 at a distance equal to the the distance between adjacent apertures42, 44 in the upper bands 46, 48 and lower band 36 on integument strip200. Still referring to FIGS. 14 and 15, it should be understood that atotal of four belts are employed in the internal loader/unloaderapparatus 210: upper studded drive belt 218a; lower studded drive belt218b; upper receiving belt 220a; and lower receiving belt 220b. Belts218a, 218b, 220a and 220b serpentine through pressure chamber 192,changing levels within the chamber 192 as dictated by the belt guidechannels 216 which are inclined as the path nears an end of pressurechamber 192.

In operation, tractor feed apparatus 50 transports the leading end ofintegument strip 200 into and through the guide of cutter 202 attachednear the entrance of pressure chamber 192 of autoclave 186. An externaldrive motor 226 has a sealed drive shaft 228a extending into pressurechamber 192 and serves to actuate rollers 232, 233 by means of gears230a, 230b and receiver shaft 228b which are supported within pressurechamber 192 and form a portion of the internal loader/unloader apparatus210 for driving the belts 218, 220. The rollers 232 and 233, in turn,actuate and rotate drive belts 218 and 220 as previously shown anddescribed with reference to the tractor feed apparatus 50. The externaldrive motor 226 will turn rollers 232, 233 and thus transport belts 218,220 at the same speed that tractor feed apparatus 50 transportsintegument strip 200 into the guide of cutter 202. Integument strip 200will thus be loaded in serpentine fashion into the autoclave 186. Whenthe autoclave 186 is loaded with integuments 22, the cutter knife 204 isactuated by controller 150 to cut the strip 200 from the continuouslength 24. After the trailing edge of integument strip 200 is loaded,controller 150 will stop the external drive motor 226. It will thenactuate the pneumatic cylinders 196 to close the closure 194 ofautoclave 186 and initiate the sterilization process. The sterilizationprocess is accomplished through conventional means such as a steamgenerator 234. Water inlet valves 236 and drain valves 238 are alsoprovided as shown in FIG. 14. Upon completion of the sterilizationprocess, the external drive motor 226 is again actuated to unload thesterilized integument strip 200 from the autoclave 186 whilesimultaneously loading a new unsterilized integument strip as justdescribed.

Because the sterilization unit 100 is a batch operation, the precedingoperations at the media fill station 70 and fill check scanner 90 mustbe halted until the sterilization unit 100 is emptied to receive a newbatch of cellules 30. Means can be provided to permit a continuousoperation such as by rolling the length 24 of cellules 30 passing fromfill check scanner 90 onto a spool or supporting the length 24 on anelongated tractor feed track until the sterilization unit 100 is readyto accept a new batch of cellules 30. A cutter, such as cutter 202,would be used to cut a length of cellules 30 for later insertion intosterilization unit 100. Such means would permit the continuous fillingof cellules 30 with media 92.

An alternative to the sterilization unit 100 includes the use of apresterilized length 24 of cellules 30 and filter sterilized media 92.Using presterilized cellules and filter sterilized media eliminates theneed for a sterlilization unit 100 in the automated system 10. Theelimination of the sterilization unit 100 permits a continuous operationfrom the fill scanner unit 90 to the cooling and storage unit 110. Apresterilized length 24 of cellules 30 may be produced since themembrane for the cellules is aseptic at the time of manufacture. Theinteguments 22 would then be produced as previously described underaseptic conditions. The filter sterilized media would be prepared andsterilized by an inline filtration process.

Cooling and Storage Unit 110

Referring now to FIGS. 16 and 16A, there is depicted the cooling andstorage unit 110 which generally comprises cooling chamber 240, an airfilter assembly 242, and cooling system 244. Air filter assembly 242includes a filter housing 246, a prefilter 248, a blower motor 250driving to a squirrel cage blower assembly 252, a flume 254 and ahepafilter 256. Air filter assembly 242 is attached to and supported bythe upper surface 258 of the cooling chamber 240. Filter housing 246includes an air intake aperture 262 which is covered by prefilter 248and attached to the housing 246. Prefilter 248 filters dust and otherlarge airborne particles and prevents them from being drawn into the airfilter assembly 242. Mounted within air filter housing 246 is thesquirrel cage blower assembly 252 which is driven by a blower motor 250mounted externally to the cooling chamber 240. Blower assembly 252 drawsair from the ambient atmosphere through the prefilter 248 and injectsthe air through flume 254 into the hepafilter 256 which covers theaperture 264 formed in the upper surface 258 of the cooling chamber 240.The hepafilter 256 removes 99.97% of all pollutants and airbornecontaminates from the air injected into the cooling chamber 240.

Cooling system 244 includes cooling coils 266 which are supported nearthe top of cooling chamber 240 on a perforated support plate 268 whichis affixed to the sidewalls and endwalls of the chamber 240 so as to beparallel with the upper surface 258 of the chamber 240. Throughconventional means, coolant is circulated through the cooling coils 266so as to maintain a constant temperature within the cooling chamber 240of five degrees centigrade.

The air drawn into the cooling chamber 240 is vented through the entryport 272 and exit port 274 for integument strip 200. A positive pressureof 1.1 atmospheres is maintained within the cooling chamber 240. Thecontinuous flow of filtered air through entry and exit ports 272 and 274resulting from the positive air pressure within cooling chamber 240prevents contaminants, such as air-borne microorganisms, from enteringthe cooling chamber 240 and contaminating the previously sterilizedmedia. As shown in FIG. 16, cooling chamber 240 includes a housingextension 270 having a front face 273 in which entry port 272 is formed.Housing extension 270 extends from cooling chamber 240 to a position inclose proximity to sterilization unit 100 so as to minimize the distancetravelled by the sterilized cellules 30 before they enter the coolingand storage unit 110. After leaving sterilization unit 100 and beforeentering cooling and storage unit 110, the sterilized cellules 30 areexposed to the unfiltered air of the ambient environment. However, afterundergoing the heat sterilization process, the heat radiating from thesterilized cellules 30 creates air currents which, along with gasesgenerated by the hot media, combine to drive away air-bornemicroorganisms which might otherwise contaminate the media 92 or thesurfaces of cellules 30 before they enter the sterile environment ofcooling and storage unit 110.

The tractor feed apparatus 50, previously described, is supported withinthe cooling chamber 240 and extends outside the enclosure through entryand exit ports 272, 274. Because the extremely high temperatures presentin the sterilization unit 100 are not present in the cooling and storageunit 110, tractor feed apparatus drive motors 112 may be located withinthe cooling chamber 240; however, to allow as many cellules 30 aspossible to be contained within the cooling chamber 240, it is preferredthat tractor feed drive motors 112 be mounted outside cooling chamber240. As previously described with reference to the sterilization unit100, integument strip 200 is supported in serpentine arrangement withincooling chamber 240 by a series of perforated support plates 276. Incooling chamber 240, the perforated support plates 276 are rigidlyattached perpendicularly to the coil support plate 268. These supportplates 276 in turn support the guide belt channels 216 and drive belts218 and 220 in a multi-level serpentine fashion as described above andillustrated in FIGS. 14-15 with regard to the sterilization unit 100. Inoperation, the leading edge of integument strip 200 is inserted intoentry port 272 to the cooling chamber 240 and is loaded therein inserpentine fashion. The sterilized cellules 30 are stored in coolingchamber 240 until the media 92 and cellules 30 are cooled. Then, asrequired, the sterile media 92 and cellules 30 of integument strip 200are drawn into the tissue manipulation unit 120 described below. Thesterilization unit 100 can sterilize one integument strip 200 at a time.However, it is desirable that cooling and storage unit 110 have thecapacity to cool and store a plurality of such integument strips 200simultaneously and to house the sterile cellules 30 until needed.

Tissue Manipulation Unit 120

Referring again to FIG. 1, the tissue manipulation unit 120 generallyhouses a cellule cutting unit 280, a tissue planting unit 290, a sealingunit 310 and a surface sterilization unit 320. In the tissuemanipulation unit 120, the sterilized cellules 30 with growth media 92from the cooling and storage unit 110 are invested with a tissue sample122. The tissue sample may be meristematic tissue from a stock or parentplant or more often is tissue from either a stage 1 initial culture or astage 2 multiplication culture grown in cellules of a previousintegument strip 300 transported from the culture room 130. The tissueplanting unit 290 will ordinarily receive plant material for investingin media-filled cellules from cellules previously housed in the cultureroom 130 and opened by cutting unit 280. However, seeds or meristematictissue may be manually fed into tissue planting unit 290 for insertinginto the media-filled cellules. As depicted in schematic form in FIG. 1and for purposes of the description below, it is assumed that thecellules of sterilized integument strip 200, previously described, areto be filled with plant tissue that has previously been grown in cultureroom 130 in an integument strip 300 comprising a plurality of cellules30 containing growing tissue 122. Integument strip 300 is transportedfrom the culture room 130 into the tissue manipulation unit 120 wherethe cellules 30 with tissue 122 first undergo surface sterilization insurface sterilization unit 320. The sterilized cellules 30 with tissueare then opened by cellule cutting unit 280 and the growing tissue 122contained therein is removed and cut into smaller tissue samples whichare then inserted into unused and sterilized cellules 30 of integumentstrip 200 in the tissue planting unit 290. The newly planted cellulesare then sealed by sealing unit 310, are coded with a bar code by barcoding means 311 and transported back to culture room 130.

Referring still to FIG. 1, the tissue manipulation unit 120 includes abox-like enclosure 282 having an air filter assembly like the onedescribed above with regard to the cooling and storage unit 110. The airfilter assembly filters the air that is used to pressurize the enclosure282, such pressurization precluding the entrance of airbornecontaminates such as microorganisms which could contaminate the cultures122 during any of the tissue manipulations which take place within thetissue manipulation unit 120. A positive pressure of approximately 1.1atmospheres is maintained in enclosure 282. An entrance port 282a to thetissue manipulation enclosure 282 is formed in one end and is sealinglyattached to the exit port of the cooling and storage unit 110 so that noairborne contaminants can enter the enclosure 282. In this manner,cellules 30 making up integument strip 200 transported from the coolingand storage unit 110 pass directly into the tissue manipulation unit 120and are continuously exposed to filtered air. Air is exhausted fromenclosure 282 through the entry and exit ports 282a, b for integumentstrip 200 and entry and exit ports 282c, d for integument strip 300.

Tractor feed apparatus 50 extends into and through enclosure 282 so asto transport sterile integument strip 200 from the cooling and storageunit 110 into the tissue manipulation unit 120 and to transport tointegument strip 200 to culture room 130 once tissue samples 122 havebeen placed in the cellules 30 from tissue-filled integument strip 300and once the cellules have been sealed. Tractor feed apparatus 50 isalso employed to transport integument strips 300 containing sealedcellules with growing tissue therein from the culture room 130 to thetissue manipulation unit 120, and to discharge used integument strips300 from enclosure 282 to disposal unit 170 after tissue samples 122have been removed from the cellules 30.

Surface Sterilization Unit 320

Referring now to FIGS. 17 and 18, after being transported from theculture room 130 to the tissue manipulation unit 120, cellules 30 ofintegument strip 300 containing living plant tissue 122 first enter thesurface sterilization unit 320. Surface sterilization unit 320 includesan enclosure 284 which is divided into three compartments 286, 287, 288that are separated by flap-like closures 292a, 292b, 292c, 292d.Closures 292 span the entire cross-section of enclosure 284 and serve toprevent solution from being sprayed or splashed out of the compartments286, 287, 288. Enclosure 284 is preferably made of acrylic plastic, suchas plexiglass, approximately 1/2 inch thick. As depicted in FIGS. 17 and18, tractor feed apparatus 50 transports cellules 30 in integument strip300 containing the living tissue 122 through slit formed in closure 292aand between a pair of sterilization spray bars 294 which are attached tolower support plate 296 which serves as part of enclosure 284.Sterilization spray bars 294 comprise plastic tubing approximately 1/2inch in diameter having perforations 298 in the sides. The lower ends ofthe spray bars 294 are connected to flexible tubing 302 through which asterilizing solution of sodium hypochloride is pumped by pump 304 from astorage tank 306. As the cellules 30 pass between the spray bar 294, thesterilization solution is sprayed on the outside surfaces of thecellules 30 through open windows formed in support plates 74, 76. Thesprayed solution then runs down the sides of the cellules 30 and iscollected in drain basin 308 in fluid communication with holding tank312 via drain line 314.

After undergoing the surface sterilization in compartment 286, thecellules 30 are then drawn through a slit formed in closure 292b andinto an identical compartment 287 where they pass between a second setof spray bars 318 which are connected to a source 322 of sterilizedwater. The sterilized water is sprayed on the cellules 30 by pump 323through open windows in support plates 74, 76 to wash away remainingsterilization solution. The resulting fluid is then collected in asecond drain basin 324 where it is drained via drain 326 to a secondholding tank 328.

Referring now to FIGS. 17 and 18A, the sterilized and washed cellules 30of integument strip 300 then pass through a slit formed in closure 292cand are drawn into a drying chamber 330 in compartment 288. Filtered airwithin tissue manipulation unit 120 is blown down and over the surfaceof cellules 30 by squirrel cage blowers 331. The air is funneled overthe surface of cellules 30 by unperforated plates 332a, 332b. Solutionwhich drips off the surface of cellules 30 is collected in basin 324 anddrained to holding tank 328 via drain line 315. Once the cellules 30have been sterilized and dried, they are transported through slit formedin closure 292d and into cutting unit 280 as shown in FIGS. 1 and 19.

Cutting Unit 280

Referring now to FIGS. 1 and 19, once in the cutting unit 280, the loweredge of integument strip 300 is drawn across stationary cutting blade334, best shown in FIG. 20A, which cuts open the bottom of the cellules30 just above lower heat seal band 36. Cutting blade 334 will be heatedto destroy any contamination which may be deposited on blade 334 due toa contaminated plant in a cut open cellule. Lower heat seal band 36 andthe attached lower portion of the cellules 30 is then transported out ofthe manipulation unit enclosure 282 by the lower drive and receivingbelts 218b, 220b of the tractor feed apparatus 50 shown in FIG. 15 fordisposal in disposal unit 170. The now open cellules 30 are nexttransported into the tissue planting unit 290.

Tissue Planting Unit 290

Referring now to FIGS. 19, 20A, and 21 tissue planting unit 290generally includes a compartmentalized and rotatable tissue containmentdevice 336, a water injection means 338 and pneumatic clamps 340.

Water and/or air injection means 338 is employed to pierce the closedend of a cellule 30 whose other end has been opened by cutting unit 280and inject water such that the water pressure and the force of gravitycauses the tissue sample 122 contained therein to pass out the bottom ofthe open cellule 30 into the tissue containment device 336. The waterinjection means 338 comprises a hypodermic-like needle 342 in fluidconnection with a sterile water source 344 and a pumping means 346. Bestshown in FIGS. 19 and 21, upon receipt of the appropriate signal fromthe controller 150, a pneumatic clamp 340 closes and clamps an opencellule 30 along heat seals 38 so as to maintain the cellule's positiondirectly above the tissue containment device 336. Controller 150 nextsignals water injection means 338 such that needle 342 is pneumaticallylowered by cylinder 350 so as to pierce the top of the opened cellule 30as shown in FIGS. 19 and 20A. The pumping means 346 is then actuated anda stream of sterile water is injected into the top of open cellule 30.As shown in FIG. 21, the injected water and gravity cooperate to depositthe tissue sample 122 in one of the compartments 352 of the tissuecontainment device 336 directly below the bottom of opened cellule 30.It should be understood that pressurized air may be used in place of thesterilized water. Once the cellule's tissue sample 122 has beendeposited in the tissue containment device 336, the used cellules aretransported out of the tissue manipulation system enclosure 282 by theupper drive and receiving belts 218a, 220a of the tractor feed apparatus50 for disposal in disposal unit 170 as shown in FIG. 19.

Referring now to FIGS. 20A and 21, the compartmentalized and rotatabletissue containment device 336 comprises a rotatable inner hub 354mounted on drive shaft 356 which is driven by a stepping motor (notshown). Rotatable hub 354 includes a plurality of flat bottom plates 355disposed around the circumference of the hub and forming a multi-sidedpolygon. On each side of hub 354 are multi-sided polygon shaped endplates 363 which extend from shaft 356 to bottom plates 355. Shaft 356passes through, but is not attached to, the circular end plates 358 andside plates 360. Identically dimensional rectangular windows 362 areformed in end plates 358 and side plates 360 and are coaxially alignedwith push rod 376 and extraction member 378 as described below.Radiating from and attached to rotatable hub 354 are a plurality ofpairs of flat spokes or divider plates 364. Each pair of spokes 364 isattached at its inner end to a bottom plate 355 of hub 354 therebyforming a compartment 352. The outer end is open to provide the openingto compartment 352. Arcuate rim segments 366 connect the outer ends ofadjacent spokes 364, but do not extend over the openings to compartments352. Bracing members 367 may be used to span the entrances tocompartments 352 and provide rigidity to the containment device 336. Inthis configuration, tissue containment device 336 comprises acompartmentalized, carousel-like, device having a plurality ofcompartments 352 defined by outer surfaces or bottom plates 355 of hub354, inner surfaces of flat spokes 364, and the inner surfaces ofstationary end plates 358. The number and size of compartments 352 maybe varied by changing the diameter and width of tissue containmentdevice 336.

The outer surface 355 of hub 354, which forms the bottom of compartments352, is provided with perforations 370a to allow the water and lessviscus media 92, which were deposited along with plant tissue 122 bywater injection means 338, to drain from compartments 352 and into hub354. Such fluids may also drain from compartments 352 by seaping betweenstationary end plates 358 and the edges of flat spokes 364 adjacentthereto. Perforations 370b are also formed in side walls 357 of hub 354to allow collected liquids to drain therethrough and to seap between hub354 and end plates 358. Such liquids may also drain from hub 354 throughperforations 370a of the lower, empty, compartments 352. All suchliquids drain basin 368 where they then drain to disposal tank 372 shownin FIG. 21.

After a cellule 30 is opened and tissue sample 122 is deposited in acompartment 352 of the tissue containment device 336, the controller 150actuates the stepping motor to turn tissue containment device 336 apreset number of degrees and to activate the tractor feed apparatus 50moving the integument strip 300 forward, so as to bring a newly openedcellule 30 directly above the next empty compartment 352 in the tissuecontainment device 336. In this step-like manner, a compartment 352containing a tissue sample 122 is positioned between the aligned windows362 in the circular end plates 358 and side plates 360. Once sopositioned, the tissue sample 122 may then be removed from thecompartment 352 and cut into a plurality of tissue samples by thecutting mechanism 371 as hereinafter described.

Cutting Mechanism 371

Referring now to FIG. 20A, the cutting mechanism 371 comprises pneumaticcylinders 374, 384, pushrods 376, 382, extraction member 378, andcutting blade 380. Extraction member 378 is a rectangular-shaped blockof stainless steel having a cross section identical in shape to thecross section of a compartment 352 of tissue containment device 336 andrectangular windows 362 in end plates 358 and side plates 360. Member378 is attached to pushrod 376 and is actuated by pneumatic cylinder374. Extraction member 378 reciprocates in a cutting channel 379 havinga cross-section which slidingly receives extraction member 378. Thecutting channel 379 extends through windows 362 in end plate 358a andside plate 360a, one of the compartments 352, windows 362 in end plate358b and side plate 360b, and exits into planting conduit 398. Cuttingblade 380 is sideably disposed within a blade guide 381 at the exit ofcutting channel 379 and is attached to pushrod 382 which is actuated bypneumatic cylinder 384.

When tissue sample 122 in compartment 352 is to be multiplied into aplurality of new samples, controller 150 will actuate cylinder 374 andpushrod 376 so that extraction member 378 is extended through cuttingchannel 379 and the compartment 352 in tissue containment device 336which is then aligned between windows 362 in end plates and side plates358, 360. As pushrod 376 is further extended as shown in FIG. 20B,extraction member 378 pushes the tissue sample 122 out of compartment352 until a portion of the tissue sample 122 extends through the exit ofcutting channel 379 beneath cutting blade 380 in blade guide 381.Pushrod 382 connected to cutting blade 380 is then actuated by pneumaticcylinder 384 so that cutting blade 380 is propelled downward and seversa portion of the tissue sample 122, the severed portion then resting onworking surface 388 within planting conduit 398. Referring now to FIG.21B, with cutting blade 380 still in its lowered position, stuffingmechanism 390 is actuated by controller 150, stuffing mechanism 390including pushrod 392, pneumatic cylinder 394 and stuffing member 396.Stuffing member 396 reciprocates in a planting conduit 398 having across-section which slidingly receives stuffing member 396. Plantingconduit 398 extends past the exit of cutting channel 379 to the open endof cellules 30 at fill gate device 400. Pushrod 392, actuated bypneumatic cylinder 394, is extended so that stuffing member 396, whichis slideably engaged with working surface 388, pushes the severed tissuesample 122 through planting conduit 398 and into a sterile media-filledcellule 30. As shown in FIGS. 19 and 21A, a fill gate device 400 such asthat described previously with respect to the media fill station 70 isattached to the end of planting conduit 398 so as to separate theopposing membrane surfaces 46, 48 of integument strip 200 and therebyfacilitate investment of the cellule 30 with the severed tissue sample122. As the sterilized integument strip 200 is transported from thecooling and storage unit 110 into the tissue planting unit 290,integument strip 200 is rotated 90° from its previous upright positionso that the cellules 30 in integument strip 200 can be invested with atissue sample 122 through fill guide 400.

The process described above is employed to multiply tissue culturesalready growing in cellules 30 of integument strip 300. When firstbeginning the micropropagation process, before initial cultures havebeen established in cellules 30 of integument strip 300, it is necessaryto establish initial cultures for later multiplication. This isaccomplished by manually inserting samples of meristimatic tissue from aselected parent plant or cultivar into the tissue planting unit 290,investing the tissue samples in cellules 30 of integument strip 200,sealing the cellules and transporting them to culture room 310.Accordingly, forming a part of the enclosure 282 of tissue manipulationunit 120 is a normally-sealed access door 281, as shown in FIGS. 20 and21. When initiating the micropropagation process, an operator opensaccess door 281 and deposits a sample of meristimatic tissue within eachcompartment 352 of tissue containment device 336 as it rotates in thecounter-clockwise direction as viewed in FIG. 21. When a compartmentcontaining a manually-inserted tissue sample becomes aligned withwindows 362 formed in end and side plates 358, 360, the sample isinvested in cellules 30 of integument strip 200 in the same manner asdescribed above. Alternatively, access door 281 may be enlarged orrepositioned, or another access door may be provided in enclosure 282,so as to allow an operator to directly invest tissue into cellules 30 ofintegument strip 200 prior to the cellules being sealed by sealing unit310, without employing tissue planting unit 290. During this manualoperation, the operator will have manual control of the tissuemanipulation unit 120.

Sealing Unit 310

Referring again to FIGS. 1 and 19, once the severed tissue sample 122has been invested into the sterile cellule 30, the cellule 30 is drawninto sealing unit 310 comprising a pair of roller heat sealers 404,thereby completely sealing the new culture from the exteriorenvironment. Once sealed, the cellules 30 are bar coded by a bar codeprinting system 311 such as the Digimark variable information lasermarker manufactured by Videojet Systems International, Inc. of Elk GroveVillage, Ill. The bar code indicates the type of plant material in thecellule and the date the plant was invested in the cellule. The cellulesare then transported into the culture room 130.

Culture Room 130

The culture room 130 comprises a room or other enclosure containing thetractor feed mechanism 50, a temperature control system 404, and alighting system 406. As described previously, the tractor feed mechanism50 will transport the integument strips 200 in a multilevel serpentinefashion within the enclosure 130. In this system, it is not necessarythat the air be filtered since the cultures have been sealed from theambient environment by sealing unit 310 in the tissue manipulation unit120 after planting. It is preferred that the tractor feed system 50 besupported by a gridwork of support brackets and channels rather than bythe perforated support plates previously described with respect to thesterilization and cooling units 100, 110 since, in this application, itis important that the light waves generated by the lighting system aretransmitted and reflected throughout the entirety of the enclosure 130.The previously described perforated support plates block too much of thelight. As shown in FIG. 22, between each serpentined row of integuments,there is a bank of fluorescent lights 408 selected and positioned so asto maintain approximately 1,000 foot-candles of light throughout theunit 130. It is preferable that the system allow the light intensity tobe varied as the cultures are transported throughout the unit 130 suchthat when desirable to cease multiplication and grow finished plantletsat the completion of the growth period in the culture unit 130, thelight intensity can be increased to approximately 3,000 foot-candles soas to harden the plant and ready it for shipment to the commercialgrower for planting in a soil medium in the greenhouse. To enhance lighttransmission within culture room 130, the walls, ceiling and floor arecovered with a highly reflective surface 410 such as a mirrored acrylicsheet.

As depicted in FIG. 1, individual lengths of plant-filled cellules areperiodically scanned in the culture room 130 by a growth detectionscanner 140 which detects the growth of the plant or tissue. One type ofgrowth detection scanner 40 is the vision system, including the 2803-CMVIM module camera adapter and 2802 line scan camera manufactured byAllen Bradley of Milwaukee, Wis. The vision system will detect, fill,size, shape, contrast and multiple shades of gray whereby the system cannot only detect plant and tissue growth but also contamination of theplant material within the cellule. Should contamination be detected, anink jet printer 140a, such as the Excel small character ink jet printermanufactured by Videojet Systems International, Inc. of Elk GroveVillage, Ill., marks the cellule containing the contaminated plantmaterial to later avoid removing the contaminated plant material fromthat cellule at the cutting unit 280. A print registration scanner 140b,such as the Smarteye color mark registration scanner manufactured byTri-Tronics Company, Inc. of Tampa, Fla., may be located at cutting unit280 to detect the reject mark so as to not remove the contaminated plantmaterial into tissue containment device 336.

A bar code reader 141 is stationed within culture room 130 near growthdetection scanner 140 for identifying the plant material, media, anddate the plant was invested in the cellule. Bar code reader 141 may be abar code scanning system such as the Skan-4100 moving beam laser scannerand Skan-D41 bar code decoder manufactured by Skan-A-Matic of Elbridge,N.Y.

If the plant material is ready for the next stage of micropropagation,the length of cellules are transported back into the tissue manipulationunit 120. If the appropriate number of tissue multiplications have beenperformed and the desired number of plantlets have been produced, thecellules are transported from the culture room to the packaging system160 where the sealed cellules are boxed for shipment.

Control System

FIG. 23 depicts a block diagram disclosing the basic organization of thecontrol system 150 for the automated system 10 for performingmicropropagation and tissue culturing. The control system 150 iscentered around master control unit 500, a programmable controller, andfour local control units, 502, 504, 506 and 508. Local control units502, 504 506 and 508, also programmable controllers, are generallydedicated to controlling and monitoring specific portions of automatedsystem 10. More specifically, local control unit 502 is generallydedicated to the media preparation and fill units 70, 80, the fill checkscanner 90, ink jet printer 91 and bar coding means 93; local controlunit 504 is dedicated to monitoring or controlling sterilization unit100 and cooling and storage unit 110; local control unit 506 monitorsand operates tissue manipulation unit 120; and local control unit 508controls activities in culture room 130. Each local control unit alsooperates the drive motors which form a part of the tractor feedapparatus 50 within its region of control.

The master control unit 500 may be a dedicated controller system where aprogrammable logic controller, such as the PLC-3 family of controllershaving up to 4096 input/output channels manufactured by Allen Bradley ofMilwaukee, Wis., may be used to control all operations with individualunits also having keyboard input for manual operation whereby separateparts of the automated system 10 can be operated separately.

Master control unit 500 and local control units 502, 504, 506 and 508are configured in a master-slave arrangement such that master controlunit 500 can monitor all the conditions and parameters sensed by thelocal control units and can coordinate the operation of the entiresystem. Additionally, master control unit 500 can assume the function ofany local control unit when it may become necessary to remove that localcontrol unit from the system so as to reprogram certain steps or performmaintenance on the local control unit.

The control provided by local control units 502, 504, 506 and 508 willnow be described in greater detail. Referring now to FIGS. 8, 9 and 23,local control unit 502 monitors signals from fill sensors 142 andactuates media mix system 510, comprising metering pumps 138, solenoidvalve 133, stirrer motor 128 and heater 132. Local controller 502 alsooperates drive motors 112 (FIG. 6) for transporting cellules 30 throughmedia fill apparatus 70 and fill check scanner 90. Local control unit502 also has responsibility for controlling and actuating mediadispensing system 516 including fill pumps 154 and transport rack 158.Local control unit 502 also monitors signals received from fill checkscanner 90 and controls bar coding means 93 and any ink jet printer 91applying reject marks.

Referring now to FIGS. 12-14 and 23, local control unit 504 is shown toactuate motor drives 520 which transport cellules 30 within and throughsterilization unit 100 and cooling and storage unit 110. Also controlledby local control unit 504 is the autoclave loading and unloading system522, including cutter 202 used to cut the continuous length 24 ofintegument roll 20 and cylinders 196 employed to close autoclave 186.Local controller 504 also monitors and actuates the sterilizationprocess 524, including the operation and monitoring of steam generator234, and controls the cooling process 526 in cooling and storage chamber110.

Referring to FIGS. 1 and 23, local controller 506 actuates motor drives528 and 530 which, respectively, transport integument strips 300 andintegument strips 200 into and out of tissue manipulation unit 120.Local control unit 506 also controls surface sterilization unit 320,cellule cutting unit 280, tissue planting unit 290, sealing unit 310 andbar coding means 311. Local control unit 506 also monitors disposal unit170 and signals an operator when the unit if full. Local control unit506 also controls any print registration scanner 95 adjacent tissueplanting unit 290.

Referring now to FIGS. 1, 22 and 23, local control unit 508 actuatesculture chamber motor control system 542, lighting system 406 andtemperature control system 404 located within culture chamber 130. Inaddition, signals from the growth detector 140 and bar code reader 544are monitored by local control unit 508. Local control unit 508 alsocontrols any ink jet printer 140a applying reject marks.

It is the function of the control system 150 to coordinate andsynchronize all operations throughout the automated system 10. Thus thecontrol system 150 sets the timing, sequence, and speed of eachoperation by receiving input signals from the apparatus located at eachoperation station and then sending output signals to such apparatus.

While the preferred embodiment of this invention has been shown anddescribed, modifications thereof can be made by one skilled in the artwithout departing from the spirit of the invention. The embodimentdescribed herein is exemplary only and is not limiting. Many variationsand modifications of the system and apparatus are possible and arewithin the scope of the invention. Accordingly, the scope of protectionis not limited by the above description, but is only limited by theclaims which follow, and that scope includes all equivalents of thesubject matter of the claims.

I claim:
 1. An automated system for culturing plant tissue, comprising:afirst length of membrane material forming a plurality of first chambershaving an open end; a second length of membrane material having aplurality of second chambers enclosing individual plant tissue; openingmeans for opening said second chambers; removal means for removing theplant tissue from said second chambers; cutting means for cutting theplant tissue into a plurality of pieces; inserting means for insertingindividual pieces of plant tissue into said first chambers; and closingmeans for closing said open ends of said first chambers.
 2. Theautomated system of claim 1 wherein said cutting means comprises:meansfor receiving the plant tissue from the second length of membranematerial; means for cutting the plant tissue; means for transporting theplant tissue from the receiving means to said cutting means; andplanting means for transporting individual cut pieces of plant tissuefrom said cutting means to one of said first chambers and planting anindividual cut piece therein.
 3. The automated system of claim 1 whereinsaid cutting means and inserting means comprise:an enclosure having afirst entry and exit for passing the first length of first chamberstherethrough and a second entry and exit for passing the second lengthof second chambers therethrough; transport means for transporting thefirst and second lengths of chambers through said enclosure; meansdisposed on said enclosure for moving air within said enclosure; meanson said enclosure for filtering the air prior to passing into saidenclosure; means within said enclosure for surface sterilizing thesecond length of chambers with plant tissue therein; means within saidenclosure for opening the second length of chambers; means within saidenclosure for removing the plant tissue from the second length ofchambers; means within said enclosure for cutting the plant tissue intosmaller pieces; means within said enclosure for investing the smallerpieces of plant tissue into the first length of chambers; and meanswithin said enclosure for closing the first length of open chambers. 4.The automated system of claim 1 wherein said closing means includes apair of roller heat sealers for applying heat to the open end of saidfirst chamber for sealing the first chambers closed.
 5. The automatedsystem of claim 1 further comprising tractor feed apparatus fortransporting said lengths of chambers throughout the automated system.6. The automated system of claim 1 further including:a first coding unitfor uniquely coding each open chamber of said first length; a secondcoding unit for uniquely coding each sealed chamber of said secondlength exiting said closing unit; and a bar code reading unit foridentifying the plant material in said sealed chambers.